Document 13309653

advertisement
Int. J. Pharm. Sci. Rev. Res., 25(2), Mar – Apr 2014; Article No. 01, Pages: 1-6
ISSN 0976 – 044X
Research Article
Simple Scheme for the Standardization of Herbal Drug: Swasanandam Gutika
Deepa.P*, Babu. G, Mohammed Noufal.K, Mumthas .M, Shanthiya.K., Sreejisha.V.K.
Devaki Amma Memorial College of Pharmacy, Chelembra, Kerala, India.
*Corresponding author’s E-mail: [email protected]
Accepted on: 24-04-2013; Finalized on: 31-03-2014.
ABSTRACT
The present study was undertaken to standardize the marketed formulation using simple, non-expensive methods. The
standardization gives total information and controls to essentially guarantee consistent composition of herbal drugs. The present
standardization involves pharmaceutical evaluation, organoleptic evaluation, and physical evaluation along with the quantitative
estimation of alkaloids, flavonoids, saponins, total phenolics etc. Quality assurance is an integral part of traditional medicine, which
ensures that it delivers the required quantity of quality medicament. This standardization helps quality control to assess safety,
efficacy and quality of herbal medicine.
Keywords: Extractive value, Ash value, total phenolic, flavonoid, saponins.
INTRODUCTION
T
he W.H.O. has been encouraging and promoting the
herbal medicines in health care programmes. Hence
the standardization of the raw drugs, processing
finished products, verification of the claims, mechanism
of action and purity from metallic and microbial
contamination were some of the major issue which have
to be taken in to consideration for increasing the world
wide acceptability of herbal products and also to achieve
clinical success and maximum therapeutic effect1.
According to an estimate of the world Health
Organization (WHO), about 80% of the world population
still uses herbs and other traditional medicines for their
primary health care needs. Herbal formulations have
reached widespread acceptability as therapeutic agents
for diabetics, arthritics, liver disease, and cough
remedies2. As per WHO definition, there are three kinds
of herbal medicines: raw plant material, processed plant
material and medicinal.
Herbal formulations have reached extensive acceptability
3
as therapeutic agents for several diseases .The
development of authentic analytical method which can
reliably profile the phytochemical composition, including
quantitative analyses of marker/ bioactive compounds
and other major constituents, is a major challenge to
scientists. Standardization is an important step for the
establishment of a constituent biological activity, a
consistent chemical profile, or simply a quality assurance
programme for production and manufacturing of herbal
drugs4. WHO mentioned specific guidelines for the
assessment of safety, efficacy and quality of herbal
medicines as a pre requisite for global harmonization
were of utmost important 5.
Standardization is a system that ensures a predefined
amount of quality and therapeutic effect of ingredients in
each dose6. Therapeutic activity of an herbal formulation
depends on its phytochemical constituents. The
development of authentic analytical methods reliably
helps to profile the phytochemical composition, including
quantitative analyses of marker or bioactive compounds.
Standardization is an important step for the
establishment of a consistent biological activity, a
consistent chemical profile or simply a quantity assurance
program for production and manufacturing of an herbal
drug7. The authentication of herbal drugs and
identification of adulterants from genuine medicinal
herbs are essential for both pharmaceutical companies as
well as public health and to ensure reproducible quality of
herbal medicine.
The subject of herbal drug standardization is massively
wide and deep. For the purpose of research work on
standardization of herbal formulations and nutraceuticals,
a profound knowledge of the important herbs found in
India and widely used in ayurvedic formulation is of
utmost importance8. India needs to explore the
medicinally important plants. This can be achieved only if
the herbal products are evaluated and analyzed using
sophisticated modern techniques of standardization. The
world health organization (WHO) has appreciated
importance of medicinal plant for public healthcare in a
developing nations and has evolved guidelines to support
the member states in their efforts to formulate national
policies on traditional medicine and to study their
potential usefulness including evaluation, safety and
efficacy.
Standardization of herbal drugs comprises total
information’s and controls to essentially guarantee
consistent composition of all herbals including analytical
operations for identifications, markers, assay of active
principles, there is no legal control model over medicinal
plants9. Different counties medicinal plants or products
derived from them in different ways and adopted
different
approaches
of
licensing,
dispensing,
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
1
Int. J. Pharm. Sci. Rev. Res., 25(2), Mar – Apr 2014; Article No. 01, Pages: 1-6
manufacturing and trading to ensure their safety, quality
and efficacy10.
Swasanandam gutika belongs to the category
bronchodilator. Chemical constituents in the formulation
consists of Shudha hingula (purified and processed
cinnabar),
Karpoora
(Cinnamomum
camphora),
Vatsanabha
(purified and processed aconitum terox),
Vara- tripala (Terminalia bellirica and Embilica officinalis).
These medicines should only be taken under strict
medical supervision. Self medication with this medicine
may prove to be dangerous. It is to be avoiding in children
and pregnant ladies, over dosage may cause many side
effects including gastritis.
ISSN 0976 – 044X
between platens, one of which moves to apply deferent
force to the tablet to cause fracture. The term crushing
strength is also frequently used to describe the resistance
of tablets to the application of a compressive load, and
the result limit is ± 5%.
Procedure
The apparatus consist of two jaws facing each other, one
of which more towards the other. Measurement was
carried out on 10 tablets, taking care to remove all the
fragments of the broken tablets before each
determination and these takes average hardness.
Limits-normal tablet hardness ranges from 3-6kg.
MATERIALS AND METHODS
2. b. Friability
Collection of materials
It is the phenomenon where the surface of the tablet is
damage shown site of damage due to mechanical shock.
Marketed ayurvedic product was
“Mangalodhayam Pharmaceuticals”
Malappuram.
collected from
Changarakulam,
Materials used
Test Tube, Conical flask, Beaker, Muffle Furnace, Crucible,
Petri dish, Funnel, Filter paper, Standard flask, Monsanto
hardness tester, Roche friabilator, Hot air oven, U.V.
Spectrophotometer, TLC plate, U.V. Chamber.
The entire tests were carried out in the laboratory of
Devaki Amma Memorial College of Pharmacy. Gutika was
taken for standardization. Organoleptic characters and
Physico chemical properties like colour, odour, taste,
texture, pH, loss on drying, total ash, acid insoluble ash,
water soluble ash, different extractive values etc were
determined. Swasanandam gutika was studied for
uniformity of weight and disintegration time.
1. Organoleptic evaluation
Organoleptic characters of the sample were carried out
based on color, shape, texture, odor, taste, pH, uniformity
of weight, average weight of gutika were evaluated (Table
1).
1. a. pH
pH of the formulations in 10% w/v of water soluble
portions was determined using standard glass electrode
at 24°C according to the prescribed standard method in
Indian Pharmacopoeia.
1. b. Uniformity of weight
11
Procedure
Accurately weighed the tablets, removed any loose dust
from the tablet as before. A maximum mean weight loss
from the 3 sample of not more than 1% was considered
acceptable for most products.
Percentage of friability of the tablets of a badge can be
found by the following formula.
Percentage friability = (W2/W1)100
W1-weight of tablets before testing
W2-weightof tablets after testing
2. c. Disintegration tests11
Disintegration test was performed in a basket rack
assembly supporting six glass tubes of 3 inches long, open
at the top and held against a 10-mesh screen at the
bottom end of the basket rack assembly. One tablet was
placed in each tube and the assembly was moved up and
down at a frequency of 30 cycles per min at a
temperature of 37°C.
2. d. Fluorescence analysis:
For fluorescence test, 1 mg of powdered drugs was
exposed to ultraviolet light at wavelength of 365 nm and
daylight while wet, after being treated with different
reagents (Table 2).
3. Physico-Chemical evaluation
Extractive values
20 Gutika of Swasanadham gutika was weighed
individually and their average weight was calculated.
2. Physical Evaluation
2. a. Hardness test 11
Hardness is also so called crushing strength. It is the load
required to crush the tablet when placed on its edge.
Measure of the mechanical integrity of tablet is their
breaking force, which the force required to cause them to
fail in a specific plane. The tablets are generally placed
This method determines the amount of active
constituents in a given amount of marketed formulation
when extracted with solvents. It is employed for that
material for which no chemical or biological assay method
exists. As mentioned in different official books the
determination of water soluble and alcohol soluble
extractives, is used as a means of evaluating crude drugs
which are not readily estimated by other means.
The extraction of any drug with particular solvents yields
a solution containing different phyto constituents. The
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
2
Int. J. Pharm. Sci. Rev. Res., 25(2), Mar – Apr 2014; Article No. 01, Pages: 1-6
composition of these phytoconstituents in those
particular solvents depends upon the nature of the drug
and solvent used. The use of a single solvent can be the
means of providing preliminary information on the quality
of a particular drug sample.
Procedure
3. a. Chloroform soluble extractive11
5gm of the air dried powder of the marketed sample was
macerated with 100 ml chloroform in a stopper flask, and
st
was shaken frequently during the 1 6hrs and was
allowed to stand for 18hrs. There after it was filtered
rapidly taking precaution against the loss of solvent. 25ml
of the filtrate was evaporated to dryness in a tarred flat
bottom shallow disk dried at 105oC, and weighed and the
percentage chloroform soluble extractive was calculated
(Table 3).
3. b. Ethanol soluble extractive11
5 gm of the air dried powder of the given marketed
sample was macerated with 100 ml ethanol in a stopper
flask and was shaken frequently during the 1st 6 hrs and
allow standing for 18hrs. There after it was filtered rapidly
taking precaution against the loss of solvent. 25 ml of the
filtrate was evaporated to dryness in a tarred flat bottom
shallow disk dried at 105ᵒC, and weighed and the
percentage of ethanol soluble extractive was calculated.
3. c. Water soluble extractive 11
5gm of the air dried powder of the given marketed
sample was added in to 50 ml boiled water at 800c in a
stopper flask. It was allowed to cool and filtered. 5ml of
filtrate was transported to a tarred evaporating dish,
other solvent was evaporated on a water bath and
allowed to dry for 30min. Finally dried in an oven for 2 hrs
and residue was weighed and the percentage of water
soluble extractive was calculated (Table 3).
3. d. Ash values
The ash of any material is composed of their non volatile
inorganic components. Controlled incineration of
marketed drug results in an ash residue consisting of an
inorganic material. This value varies with in fairly wide
limits and is there for an important parameter for the
purpose of evaluation of the formulation. In certain
formulation, the percentage variation of the weight of ash
from sample to sample is very small and any marked
difference indicates a change in quality. Unwanted parts
of drugs, posses a character that will raise the ash value.
More direct contamination such as by sand or earth is
immediately detected by the ash value. The ash value can
be determined by 3 different methods to measure the
total ash, acid insoluble ash, water soluble ash (Table 3).
Procedure
3. d. 1. Total ash11
Weighed accurately a quantity of the powdered ayurvedic
tablet equivalent to 1 gm in a tarred platinum dish and
ISSN 0976 – 044X
o
incinerated to a temperature not exceeding 550 C until
free from carbon, approximately for about 4 hrs, cooled
and weighed. The percentage of ash was calculated.
7,10
3. d. 2. Sulphated ash
Silica or platinum crucible was made to redness for
10mins and allowed to cool in a desiccator and weighed.
One gram of the substance accurately weighed was put in
to crucible and ignited gently at first. Cooled, moistened
the residue with 0.5ml of sulphuric acid, heated at 550oC
for about 4hrs. Again cooled and weighed. Percentage of
ash was calculated.
3. d. 3. Acid insoluble ash7,10
Powdered ayurvedic tablet equivalent to 1gm of the air
dried drug was accurately weighed in a tarred platinum
dish and incinerated to a temperature not exceeding
o
550 C until free from carbon, approximately for about
4hrs, cooled and weighed. The percentage of ash was
calculated. The total ash obtained by the above method
was boiled for 5mints with 25ml of 2M hydrochloric acid
and filtered through an ash less filter paper. The filter
paper was ignited in the silica crucible, cooled and then
acid insoluble was weighed.
4. Pharmacognostical evaluation
Phytochemical screening was carried out in order to find
the various constituents present in the herbal drug. The
drug in methanol was tested for the presence of alkaloids,
glycosides, steroids, flavonoids, tannins, phenolic
compounds, proteins and carbohydrates.Table:4.
4. a. Heavy metals
Limit tests were performed as per WHO prescribed
procedure.
4. b. Presence of foreign organic matter
Herbal drugs should be made from the stated part of the
plant and be devoid of other plants of the some plants or
the other plants. They should be entirely free from
moulds or insect, including excreta and visible
contaminant such as sand and stones, poisonous and
harmful foreign matter and chemical residues. Animal
matters such as insects and invisible microbial
contaminants, which can produce toxins, are also among
the potential contaminants of herbal medicines.
Macroscopic examination can easily employ to determine
the presence of foreign matter, although microscopy is
indispensable in certain special cases.
Procedure
100 grams of sample was weighed and spreaded the
sample on a white tile or a glass plate uniformly without
overlapping. The sample was inspected with naked eyes
or by means of lens (5x or above). Foreign organic matter
was separated manually. After complete separation, the
matter was weighed and %weight by weight present in
the sample was determined.
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
3
Int. J. Pharm. Sci. Rev. Res., 25(2), Mar – Apr 2014; Article No. 01, Pages: 1-6
4. c. Moisture content
The determination of moisture in a substance is done for
many purposes such as assessment of quality, quality
control, quality assurance, detection and estimation of
adulteration. The reproducibility results can be obtained
under a set of standardized condition .The activity of any
product depends on
1) Chemical composition of substance
2) The state of aggregation of its constituents
3) Water content
Procedure11
1) The substance was placed and its weight was taken
before measuring the moisture content.
2) The oven was heated to 200 degree for 3 hrs.
3) The substance was removed and weighed.
4) The drying of the substance was repeated at 200
degree in the interval and the substance was weighed
between times until the last two weights remaining same
indicating that the substance had reached constant mass.
5) The constant mass amount was subtracted from the
weight before drying to determine the weight of moisture
evaporated from the substance.
6) The weight of moisture in the substance was divided by
the weight before drying and multiplied by 100 to
determine the % of the substance.
4. d. TLC finger printing profile
Preparation of extract of Swasanandam Gutika
Refluxed 1gm of powdered drug with 25 ml methanol for
2hrs and filtered. Extracted further with another 25ml
methanol for 1hr and again filtered. Evaporated the
combined filtrate to dryness. Dissolved the residue in
25ml Methanol.
TLC study of methanolic extracts of the individual
ingredients, in Swasanandam Gutika (marketed
formulation) were carried out along with the different
marker compounds corresponding to the active
ingredients. It ensured the presence of active ingredients
.
in the formulations
ISSN 0976 – 044X
using a capillary tube by keeping 1-3cm from the bottom
of the TLC plate. The portion of the sample spots were
marked.
When the spot has been dried, the plate was placed in
the chamber at angle of 45 degree. Here ascending
development technique was used. It is important in TLC.
Due to that the development chamber was perfectly
saturated with the mobile phase. The solvent raised by
capillary action dissolved the sample mixture into discrete
spot. After that plate was removed from the tank. Thus
solvent was marked and plate was dried
The separated spots were identified by various physical
methods, such as UV light and chemical method such as
suitable reagent known as visualizing or spraying reagent.
After the location of separated compounds, the next job
is their identification. This was done by calculating Rf
values of different compounds.
Rf value = Distance travelled by solute from baseline
Distance travelled by solvent
Procedure
The plate used was having 2cm width and 12cm in length.
1) Preparing of slurry
100gm of silica gel was weighed and mixed with 90 ml of
water to produce slurry. The prepared slurry was poured
on plate and spreaded all over the area of the plate. Dried
and kept it in a hot air oven for 10 minutes.
2) Development and detection
The plate was placed in mobile phase
The mobile phase was Glacial acetic acid: Ethyl acetate:
Diethyl ether in the ratio of 7:2:1.
When the mobile phase came to a particular length 1cm
below top, removed and passed iodine vapor. Then the
plate was taken and measured the distance travelled by
the solved front and solute by scale and the Rf value was
calculated. Rf values of sample and markers were
compared.
5. Quantification of ingredients by simple spectral
analytical technique
5. a. Total phenolic content
Thin layer chromatography is usually abbreviated as TLC.
It is a form of solid, liquid, adsorption chromatography in
which the stationary phase is thin layer of fine adsorbent
evenly spreaded over a surface of a glass plate and
mobile phase is flowing organic liquid. The principle
underlying is adsorption.
Total phenolic content of the sample was measured as
gallic acid equivalent.
In TLC the preparation was carried on a glass or glass
plate which was coated with thin uniform layer of inert
adsorbent such as silica or alumina. The plates were
activated by removing the liquid associated with the thin
layer as completely as possible. This could be done by
drying thin plate. This made the adsorbent layer active
.The solution of sample and markers were applied by
From the stock solution 10-50 microgram per ml were
prepared
Preparation of Standard solution:
1 gm of the gallic acid dissolved in 1000 ml of distilled
water, which was the stock solution.
Preparation of sample solution:
Sample scratched from the TLC was mixed with 5 ml of FC
reagent and allowed to stand at room temperature for 5
minutes and then added 4 ml of 1 M sodium bicarbonate
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
4
Int. J. Pharm. Sci. Rev. Res., 25(2), Mar – Apr 2014; Article No. 01, Pages: 1-6
ISSN 0976 – 044X
to the above mixture. After 10 minutes the absorbance
was measured at UV spectrophotometer. A calibration
curve was plotted. Total phenolics were quantified by the
calibration curve obtained by measuring the absorbance
of known concentration of Gallic acid (Table 5).
The ash value 2.07% (sulphate ash), 5.1% (total ash)
respectively which indicate that the value lies within the
range. Qualitative phytochemical studies proved the
absence of carbohydrate, proteins, amino acids and
glycosides.
5. b. Total flavonoid content
With Dragendroff reagent the drug shows a dark blue
color in the visible reagent. In presence of sodium nitrite
and sulphuric acid Dragendroff followed an intense dark
blue color and the color persists for more than 10
minutes.
Total flavonoids were measured as quercetin equivalents.
Preparation of Standard solution:
1 gm of the quercetin dissolved in 1000 ml of distilled
water, which was the stock solution.
From the stock solution 10-50 microgram per ml were
prepared
Preparation of sample solution:
To the sample scratched from the TLC , 0.2 ml of 10 %
Aluminum chloride reagent,0.2 ml of potassium acetate
were added and absorbance was recorded at 430 nm
against the blank. The amount of flavonoid was calculated
with reference to quercetin from the calibration curve
(Table 5).
Thin layer chromatography
Aconitine (marker) shows the Rf value of 0.72 complies
with the Rf value 0.69 of the sample, 1,8 cineole shows
the Rf value of 0.39 complies with the Rf value 0.41 of
the sample, Gallic acid shows 0.65 which resembles to
0.70 of Swasanandham gutika.
Table 1: Organopleptic Evaluation
Sl. No
Characteristics
Color
Brown
Shape
Round
5. c. Total alkaloid content
Texture
Hard
Total alkaloids were measured as aconitine equivalents.
Odor
Pungent
Preparation of Standard solution:
Taste
Bitter
Uniformity of weight
Complies as per I.P reference.
Avg .w t
400 mg
pH
Acidic
1 gm of the aconitine was dissolved in 1000 ml of distilled
water, which was the stock solution.
From the stock solution 10-50 microgram per ml was
prepared.
Preparation of sample solution:
Table 2: Flouresence analysis
Reagents
Results
Sample in methanol was treated with2 ml of 0.5%
dragendroff reagent and was allowed to stand for 10
minutes and the absorbance was read at 438 nm against
the blank. The amount of alkaloid was calculated with
reference to aconitine from the calibration curve (Table
5).
With Dragendroff reagent in visible
+
Dragendroff reagent followed by sodium nitrite in
visible
+
Dragendroff reagent +sulphuric acid in visible
+
10 % Sulphuric acid in UV 365 nm
-
RESULTS AND DISCUSSION
Table 3: Results of Physical, Physico-Chemical and
pharmacognostical evaluation
Tablets must be able to withstand the rigors of handling
and transportation experienced in the manufacturing
plant, in the drug distribution system and on the field at
the hands of the end users (patient or consumers).
The value of percentage friability was found to be 0.695%,
which indicates that the value lies within the range.
Purpose of friability is to evaluate the ability to the tablets
to withstand the breakage during the transportation and
handling.
Characters
Results
Hardness test
3.5 kg/cm
Friability
0.695 %
Disintegration test
complies with I.P
Water soluble extractive
5.6%
Chloroform soluble extractive
3.26
3
Methanol soluble extractive
2.84
Moisture present in the formulation leads to degradation
process. The moisture content was found to be 0.22%
Sulphated ash value
2.07 %
Total ash
5.1%
The extractive value were found to be 3.2 % (chloroform
extract), 2.8 % (ethanol extract) and 5.6% (water soluble
extract).
Acid insoluble ash
4.85
Moisture content
0.22 %.
% of foreign matter
nil
Heavy metals
nil
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
5
Int. J. Pharm. Sci. Rev. Res., 25(2), Mar – Apr 2014; Article No. 01, Pages: 1-6
Table 4: Results for preliminary phytochemical studies
Phyto constituents
Report
Carbohydrates
-
proteins
-
Amino acids
-
steroids
+
Triterpenoids
+
Glycosides
-
Flavonoids
+
Alkaloids
+
Tannins & phenolic compounds
+
Table: 5. Quantification of ingredients in the marketed
sample.
Parameters
Inference
Total Phenolics
12±7 mg/g
Total Alkaloids
45±10 mg/g
Total Flavonoid
32±2 mg/g
ISSN 0976 – 044X
REFERENCES
1. Sagar Bhanu PS, Zafar R, Herbal drug standardization, The
Indian Pharmacist, 4(35), 2005,19 - 22.
2. Amit J, Sunil C, Phytosomes: A, revolution in herbal drugs,
The Pharma Review, 2007, 11-13.
3. Patel PM, Patel NM, Goyal RK , Quality control of herbal
products, The Indian Pharmacist, 5(45), 2006, 26-30.
4. Vaidya ADB, Devasagayam TPA, Current status of herbal drugs
in India: An overview, J Clin Bio chem, 41(1), 2007, 1-11.
5. Yadav NP, Dixit VK, Recent approaches in herbal drug
standardization, Int J Integr Biol, 2, 2008,195-203.
6. Patra KC, Pareta SK, Harwansh RK, Traditional approaches
towards standardization of herbal medicines-A review, J Pharm
Science Techno, 2 (11), 2010,372-379.
7. Indian Herbal Pharmacopoeia, Indian Drug Manufacturers
Association, Mumbai, 2002.
8. British Herbal Pharmacopoeia, British Herbal Medicine
Association, 1996.
9. Quality Control Methods for Medicinal Plant Materials, WHO,
Geneva, 1996.
10.
National Health Policy, Ministry of Health and family
Welfare, Government of India, New Delhi 1983.
CONCLUSION
Physical, phytochemical and the pharmacognostic study
have been useful for deciding the identity, purity and
strength of the marketed formulation. The present study
provides a brief report on herbal drug swasanadhi gutika.
The standardization helps to build a monograph of the
Ayurvedic formulation in the Indian Formulary. This study
need to be continued.
11. Sharma AK, Gaurav SS, A rapid and simple scheme for the
standardization of polyherbal Drugs , Int J Green pharm, 2009,
134-140.
Source of Support: Nil, Conflict of Interest: None.
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
6
Download